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United States Patent |
6,225,462
|
Berry
,   et al.
|
May 1, 2001
|
Conjugated polysaccharide fabric detergent and conditioning products
Abstract
A polysaccharide conjugate comprises a polysaccharide with an attached
entity having a molecular weight of at least 5000, the polysaccharide
conjugate being capable of binding to cellulose. Preferred polysaccharides
include tamarind seed xyloglucan, locust bean gum and enzyme modified
guar. The attached entity is suitably a protein such as an enzyme,
antibody or antibody fragment, or a particle possibly having a benefit
agent such as a fragrance associated therewith. Because the polysaccharide
conjugate binds to cellulose, which is present in cotton and other
fabrics, paper, etc., binding of the conjugate to cellulose brings the
attached entity into close proximity to a surface of or containing
cellulose. The invention thus enables targeting of attached entities to
such surfaces. The invention also provides a product incorporating the
polysaccharide conjugate of the invention. The product is conveniently a
laundry product such as a fabric washing product, e.g. a detergent
product, or a fabric conditioning product. In this case the attached
entity may be an enzyme, a particle bearing fragrance, etc. The invention
also provides a method of targeting binding of an entity to cellulose by
use of the polysaccharide conjugate of the invention.
Inventors:
|
Berry; Mark John (Sharnbrook, GB);
Davis; Paul James (Sharnbrook, GB);
Gidley; Michael John (Sharnbrook, GB)
|
Assignee:
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Lever Brothers Company, a division of Conopco, Inc. (New York, NY)
|
Appl. No.:
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229043 |
Filed:
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January 12, 1999 |
Foreign Application Priority Data
Current U.S. Class: |
536/123.1; 435/12; 435/18; 435/27; 435/28; 530/300; 530/345; 530/350; 536/55.1; 536/56; 536/112; 536/123.12; 536/124 |
Intern'l Class: |
C07H 001/00; C07H 003/00 |
Field of Search: |
435/7.2,7.21,12,18,27,28
530/300,345,350
536/45,46,56,81,112,1.11,123.1,123.12,124,55.1
252/8.6,174.12,174.17,DIG. 12
|
References Cited
U.S. Patent Documents
2949397 | Aug., 1960 | Werner et al.
| |
3297604 | Jan., 1967 | Germino.
| |
3516941 | Jun., 1970 | Matson.
| |
4075405 | Feb., 1978 | Takahashi et al.
| |
4234627 | Nov., 1980 | Schilling.
| |
4681806 | Jul., 1987 | Matkan et al.
| |
5051305 | Sep., 1991 | Whitaker, Sr.
| |
5336506 | Aug., 1994 | Josephson et al. | 424/488.
|
5773227 | Jun., 1998 | Kuhn et al. | 435/7.
|
5874308 | Feb., 1999 | Kilburn et al. | 530/350.
|
6048715 | Apr., 2000 | Haynes et al. | 435/179.
|
Foreign Patent Documents |
95/34628 | Dec., 1995 | WO.
| |
Other References
Derwent Abstract of JP 61155307--published Jul. 15, 1986.
Derwent Abstract of JP 61133299--published Jun. 20, 1986.
|
Primary Examiner: Wilson; James O.
Attorney, Agent or Firm: Mitelman; Rimma
Claims
What is claimed is:
1. A laundry detergent or a fabric conditioning product comprising a
polysaccharide conjugate comprising a polysaccharide with an attached
protein, said protein being selected from the group consisting of an
enzyme, antibody, and an antibody fragment said protein having a molecular
weight of at least 5,000 Daltons , said polysaccharide having a 1-4 linked
glycan backbone structure and said polysaccharide conjugate being capable
of binding to cellulose in fabric.
2. A product according to claim 1, wherein the polysaccharide has a glucan
backbone, a mannan backbone or a xylan backbone.
3. A product according to claim 1, wherein the polysaccharide is selected
from the group consisting of xyloglucans, glucomannans, mannans and
galactomannans.
4. A product according to claim 1, wherein the polysaccharide is selected
from the group consisting of tamarind seed xyloglucan (TXG), pea
xyloglucan, low galactose galactomannans, enzyme modified guar (emg), tara
galactomannan and cassia galactomannan.
5. A product according to claim 1, wherein the polysaccharide has side
chain galactose residues susceptible to oxidation by galactose oxidase.
6. A product according to claim 1 wherein the enzyme is an oxidase,
peroxidase, catalase or urease.
7. A product according to claim 1, whrein the protein is chemically
attached to the polysaccharide.
8. A product according to claim 1, wherein the protein has available amino
groups and is chemically linked to aldehyde groups formed on the
polysaccharide.
9. A product according to claim 1, wherein a protein entity is chemically
linked to a polysaccharide selected from the group consisting of tamarind
seed xyloglucan, locust bean gum, enzyme modified guar, via aldehyde
groups produced by enzymic oxidation of galactose side chains.
10. A method of binding of a protein to cellulose by use of a
polysaccharide conjugate comprising: 1) providing a product in accordance
with claim 1; 2) providing cellulose; and 3) binding said protein to said
cellulose.
Description
FIELD OF INVENTION
This invention relates to binding of polysaccharides and concerns a
cellulose-binding polysaccharide conjugate, products including the
polysaccharide conjugate, and targeting methods using the polysaccharide
conjugate. In the context of the invention the term "polysaccharide" is
intended to cover polysaccharides and oligosaccharides, and references to
"polysaccharide" and "polysaccharide conjugate" should be construed
accordingly. The term "conjugate" is used to refer to units bound or
secured together (physically and/or chemically), with a "polysaccharide
conjugate" comprising a polysaccharide bound or secured to another entity.
BACKGROUND TO THE INVENTION
It is known that various naturally occurring polysaccharides such as pea
xyloglucan, tamarind seed xyloglucan, etc. bind to cellulose by a
polysaccharide:polysaccharide interaction; indeed this binding ability is
important in the functioning of plant cell walls.
U.S. Pat. No. 3,297,604 concerns polymer compositions containing galactose
oxidized to form a carbonyl group at the C6 position. The active carbonyl
group can react in known manner, e.g. to form cyano hydrins, bisulfite
addition compounds, oximes, hydrazones, etc. The compositions can also act
to cross-link polymers, including cellulose. The polymer, may be, e.g.,
guar, locust bean gum, etc. There is no disclosure of a polysaccharide
conjugate with attached entity of molecular weight of at least 5,000.
While the polymer composition itself may be capable of binding to
cellulose, this is not unexpected, and there is no disclosure of a
polysaccharide conjugate that is capable of binding to cellulose.
U.S. Pat. No. 2,949,397 concerns use of mineral filler coated, at least
partially, with water-dispersed organic colloid, to promote retention of
filler in cellulose fibres in paper making. The colloid may be e.g. a
galactomannans, or substituted mannan such as locust bean gum and guar
gum. The coated filler is attracted to cellulose fibres by electrostatic
action. The filler and colloid are mixed together, but separate on
standing and hence are in the form of a simple mixture not a
polysaccharide conjugate.
The paper by Hayashi et al entitled "Pea Xyloglucan and Cellulose" in Plant
Physiol. (1987) 83, 384-389 describes investigations of binding of pea
xyloglucan to cellulose, using fluorescein-labelled xyloglucan prepared by
treating xyloglucan with CNBr and incubating with fluoresceinamine, and
also using radioiodinated xyloglucan prepared by reaction of 125, with the
fluorescein moiety on xyloglucan. These labels were used to trace the
binding of the polysaccharide and are among the smallest molecular label
entities known.
The present invention is based on the surprising discovery that
polysaccharides with much larger attached entities than those used by
Hayashi et al can still bind rapidly with high efficiency to cellulose by
polysaccharide:polysaccharide interaction. This is surprising because
binding occurs at multiple sites along the backbones of the
polysaccharides, rather than at a single binding site as with
antibody-antigen interactions, and it would have been predicted that
binding would have been disrupted by the attachment of large entities to
cellulose-binding polysaccharides. The invention thus opens up the
possibility of using polysaccharides to target attached entities to
cellulose, e.g. in fabric, paper, etc.
SUMMARY OF THE INVENTION
In one aspect the present invention provides a polysaccharide conjugate
comprising a polysaccharide with an attached entity having a molecular
weight of at least 5000, the polysaccharide conjugate being capable of
binding to cellulose.
The polysaccharide conjugate is preferably capable of binding to cellulose
by polysaccharide:polysaccharide interaction.
The polysaccharide may be one that binds naturally to cellulose or has been
derivatised or otherwise modified to bind to cellulose. The polysaccharide
may be naturally occurring or synthetic.
The polysaccharide desirably has a 1-4 linked .beta.-glycan (generalized
sugar) backbone structure, which is stereochemically compatible with
cellulose, such as a glucan backbone (consisting of .beta. 1-4 linked
glucose residues), a mannan backbone (consisting of .beta. 1-4 linked
mannose residues) or a xylan backbone (consisting of .beta. 1-4 linked
xylose residues). Suitable polysaccharides include xyloglucans,
glucomannans, mannans, galactomannans, .beta.(1-3), (1-4) glucan and the
xylan family incorporating glucurono-, arabino- and glucuronoarabinoxylan.
See "Physiology and Biochemistry of Plant Cell Walls" (1990) by C. Brett
and K. Waldron for a discussion of these materials.
The minimum chain length requirement for cellulose oligomers to bind to
cellulose is 4 glucose units. For xyloglucans, the side chains make the
binding less efficient and 12 backbone glucose units (i.e. about 25 total
sugar units) are required for binding to cellulose. Structural
considerations suggest galactomannans are intermediate in binding
efficiency, and about 6 to 8 backbone residues are expected to be required
for binding to cellulose. The polysaccharide should thus have at least 4,
and preferably at least 10, backbone residues, which are preferably
.beta.1-4 linked.
Naturally occurring polysaccharides that bind rapidly and strongly to
cellulose by polysaccharide:polysaccharide interaction include xyloglucans
such as pea xyloglucan and tamarind seed xyloglucan (TXG) (which has a
.beta. 1-4 linked glucan backbone with side chains of a-D xylopyranose
and--D-galactopyranosyl-(1-2)-.alpha.-D-xylo-pyranose, both 1-6 linked to
the backbone:see Gidley et al Carbohydrate Research, 214 (1991) 200-314
for a discussion of the structure of tamarind seed polysaccharide); and
galactomammans, particularly low galactose galactomannans, such as locust
bean gum (LBG) (which has a mannan backbone of .beta. 1-4 linked mannose
residues, with single unit galactose side chains linked 1-6 to the
backbone), enzyme modified guar (EMG) (guar gum has the same structural
units as LBG but has a much higher level of galactose substitution, to the
extent that there is not enough accessible mannan backbone through which
to bind cellulose. EMG is produced by enzymic removal from guar gum of a
controllable percentage of the galactose residues to produce a range of
materials that are capable of binding to cellulose, but are cheaper and
more consistently available than LBG. See Bulpin et al. in Carbohydrate
Polymers 12 (1990) 155-168 for a discussion of EMG), tara glactomannan and
cassia galactomannan. These materials are commercially available and thus
provide potentially useful sources of suitable polysaccharides. These
materials have the advantages of being relatively cheap, and already being
accepted for food use.
The polysaccharide desirably has side chain galactose residues susceptible
to oxidation by galactose oxidase, for production of an aldehyde group for
coupling of a protein entity, as will be described below. TXG, LBG and EMG
have such galactose residues.
The attached entity may be selected from a wide range of entities that
generally perform a useful function in proximity to cellulose, e.g. in
fabric, paper, etc.
For example, the entity may be a protein, such as an enzyme, antibody or
antibody fragment.
The enzyme is conveniently an oxidase, peroxidase, catalase or urease.
These enzymes work by their substrate diffusing to them, and generate a
flux of active product of molecules that diffuse away. Redox enzymes, e.g.
oxidases such as glucose oxidase, generate hydrogen peroxide which can act
as a bleach. Peroxidase catalyses the oxidation by hydrogen peroxide of a
number of substrates. Urease catalyses hydrolysis of urea, releasing a
flux of ammonium ions which raises the local pH. Catalase is an
oxidoreductase that catalyses conversion of hydrogen peroxide to water and
oxygen.
Antibodies or antibody fragments may, for example, be used in separation or
purification techniques, as is described below, or in immunoassays.
The attached entity may alternatively be a particle, e.g. of silica,
organic polymer, etc. Such particles may have a benefit agent such as a
dye, fragrance (or perfume), cosmetic, etc. associated therewith, e.g. by
adsorption, impregnation or encapsulation.
Absorption of a benefit agent such as a perfume by particles can be brought
about simply by bringing the agent and the particles into contact, and
allowing them to stand. The benefit agent, e.g. perfume, molecules can
enter the particles by diffusion.
An alternative to the use of solid particles is to form hollow capsules in
which a shell encapsulates the benefit agent.
One approach to the preparation of microcapsules of a benefit agent such as
perfume is to disperse droplets of the benefit agent in an aqueous phase
which contains water soluble polymer, and then form a polymer shell around
these agent droplets by coacervation of the polymer at the interface
between the agent and the aqueous phase. Once formed, the capsule wall
usually requires further treatment to strengthen it. The encapsulation of
perfume by coacervation has been described by Meyer, A in Chimica, 46, 101
(1992) and in U.S. Pat. No. 5,051,305.
A second approach to the formation of microcapsules of benefit agent is to
disperse agent droplets in an aqueous phase, and then bring about a
polymerisation reaction at the interface between the droplets and the
aqueous phase. The polymerisation reaction which has mostly been employed
is the formation of an aminoplast resin. This has been used for perfume
encapsulation, as disclosed in U.S. Pat. No. 4,681,806. A typical
procedure for the production of aminoplast resin capsules enclosing
perfume is set out in U.S. Pat. No. 4,234,627, which refers back to U.S.
Pat. No. 3,516,941.
A further possibility is to form solid polymer particles, absorb the
benefit agent such as perfume into these, and then encapsulate these
particles.
Further information on encapsulation techniques is given in Risch, S. J.,
Reineccius, G. A. (Ed), "Encapsulation and controlled release of food
ingredients", ACS symposium series 590, Washington D.C., 1995. It is to be
noted that not all of the encapsulation techniques described in this
reference are necessarily suitable for the preparation of particles for
use in this invention. For instance, spray drying, which is the most
widely used encapsulation technique, generally produces water-soluble
particles which may not be particularly suitable. However, the person
skilled in the art will readily be able to select suitable techniques.
The benefit agent typically constitutes between 1 and 90% of the total
weight of the particle, and preferably constitutes at least 5% by weight
to be commercially attractive. Using encapsulation techniques, benefit
agent loadings of up to about 70% can be achieved, while absorption
techniques, e.g. using highly absorbing silicas, can achieve loadings of
up to about 90%.
The particle itself may constitute a benefit agent, e.g. silicone oil
droplets.
The benefit agent conveniently comprises perfume (also referred to as
fragrance). Perfumes are used to provide a pleasing fragrance to products
in or on which they are used, and are known in the art to be mixtures of
fragrance materials such as are discussed for example, in S. Arctander,
Perfume and Flavor Chemcials (Montclair, N.J., 1969), in S. Arctander,
Perfume and Flavor Materials of Natural Origin (Elizabeth, N.J., 1960) and
in "Flavor and Fragrance Materials--1977", Allured Publishing Co. Wheaton,
Ill. USA.
The particles suitably have a diameter in the range 0.5 to 100 microns. The
lower end of this range (0.5 to 5 microns) covers small colloidal
particles and molecular complexes.
The entity may be attached to the polysaccharide by a range of physical or
chemical means. For example, proteins are conveniently chemically linked
to polysaccharides having galactose side chains by enzymically oxidising
the galactose, e.g. using galactose oxidase, to produce an aldehyde group
to which an amino group of a protein can be chemically linked. As noted
above, TXG, LBG and EMG have suitable galactose side chains. For
polysaccharides not having suitable galactose side chains, different
methods of chemical linking of proteins can be used. Alternative
techniques include limited periodate oxidation, which requires the
polysaccharide to have two adjacent hydroxyl groups in cis orientation,
and results in the production of aldehyde groups which can be reductively
aminated. A further possibility is reaction with cyanogen bromide (CNBr)
which inserts into sugar rings at vicinal diols, both in the backbone and
side chains, to provide an isourea linkage to the amino groups of
proteins. It is preferred to use chemical techniques that do not affect
the polysaccharide backbone length, which would reduce the
cellulose-binding capability of the polysaccharide.
Polysaccharide is conveniently physically attached to particles, e.g. by
adsorption. For example, porous silica particles have surface properties
that enable firm adsorption of polysaccharide. Chemical attachment
techniques may also be used. For example, for particles carrying surface
amino groups, attachment can be by the techniques discussed above for
attachment of proteins, e.g. via oxidation of galactose side chains. For
particles with surface carboxyl or hydroxyl groups, other known forms of
chemical linkage may be used. As a further possibility, where the particle
is a liposome or micelle, hydrophobic tails fixed to the polysaccharide
can be inserted therein.
Because the polysaccharide conjugate binds to cellulose, which is present
in cotton and other fabrics, paper, etc., binding of the conjugate to
cellulose brings the attached entity into close proximity to a surface of
or containing cellulose. The invention thus enables targeting of attached
entities to such surfaces. This targeting function is of use in a number
of different potential applications including the following:
1. Targeting of enzymes to bind fabric, for example soluble oxidising
enzymes such as glucose oxidase. Such enzymes will act to release hydrogen
peroxide which can act as a bleach and thus has a fabric cleaning effect
or acts to block dye transfer during washing to prevent colour running or
greying of whites. Polysaccharide-oxidase conjugates thus find use in
treatment of new cloth, particularly cotton-containing cloth, and as an
ingredient in laundry products, such as fabric washing and conditioning
products.
2. Targeting of particles containing a benefit agent to bind to fabric. The
benefit may be, e.g., an enzyme as discussed, a fragrance, dye, cosmetic
ingredient, etc. that can to advantage be attached to fabric via the
polysaccharide. The benefit agent may be adsorbed, impregnated or
encapsulated in the particle. Polysaccharide-particle conjugates of this
sort thus find use as ingredients in laundry products.
In a preferred embodiment, the particles are porous and contain benefit
agents in the pores: such benefit agents could be dyes, fragrances,
sunscreens, etc. This embodiment involves filling the pores of the
particles with the benefit agent and then blocking the pores with a
coating of the polysaccharide so that the benefit agent does not come out
of the particle again easily. It may, however, be possible to effect agent
release if desirable, e.g. by ironing. Moreover, the coating has the
combined effect of sealing the benefit agent in the pores and giving the
particles a specific affinity for cellulose or cellulose-containing
surfaces.
3. Targeting of enzymes, antibodies, particles, etc. to bind paper, for
example to produce bleaching or dyeing of the paper.
4. Immobilising antibodies, enzymes or other molecules on a
cellulose-containing surface, e.g. cellulose particles or paper, for
instance for use in diagnostic tests or immunosorbent systems.
5. In separation or purification techniques, involving passage of
polysaccharide conjugate through a cellulose bed to remove the conjugate.
For example, by use of a suitable antibody-polysaccharide conjugate,
antigen may become bound to the conjugate and then removed, e.g. from
solution, by binding of the conjugate (and attached antigen) to cellulose,
e.g. in a cellulose bed.
An additional benefit of the invention arises from the fact that, unlike
most other targeting molecules, cellulose-binding polysaccharides are
especially robust. Proteins such as cellulose binding domain can be
inactivated (denatured) by heat or aggressive surfactants, while
polysaccharides such as LBG, TXG, etc. are completely unaffected by such
treatments. The polysaccharide conjugates of the invention thus offer the
considerable advantage of extra stability and product compatibility
compared with other targeting molecules.
In a further aspect, the present invention provides a product incorporating
a polysaccharide conjugate in accordance with the invention. The product
is conveniently a laundry product such as a fabric washing product, e.g. a
detergent product, or a fabric conditioning product. In this case the
attached entity may be an enzyme, a particle bearing fragrance, etc.
The invention also finds application in personal products, e.g. for
targeting fragrance to bind to clothes. Other applications include, for
example, diagnostic test systems, paper products etc.
The product may otherwise be of generally conventional formulation, as is
well known to those skilled in the art. For a discussion of known
detergent compositions see, for example, WO-A-95/34628, particularly pages
11 to 15.
The present invention also provides a method of targeting binding of an
entity to cellulose by use of a polysaccharide conjugate in accordance
with the invention.
The invention will be further described by way of illustration, in the
following Examples.
EXAMPLE 1
Conjugation of Locust Bean Gum with Glucose Oxidase.
Materials and Methods
Purification of Locust Bean Gum
Locust bean gum (LBG) (supplied by Meyhall) was purified according to the
following method. A 1% (w/v) LBG aqueous solution was prepared by
dissolving 15 g of LBG in 1500 ml water at 80-90 C for 30 minutes with
mechanical stirring in a Silverson Homogeniser. The resulting solution was
centrifuged at 27,000 g (Sorval RC5C, 6.times.250 ml solutions, GSA rotor)
for 30 minutes. The supernatant was removed and the remaining pellets were
dissolved in 500 ml deionised water, again at 80-90 C for 30 minutes with
the aid of the Silverson stirrer. This solution was further centrifuged as
above. The resulting combined supernatants were then precipitated in
iso-propanol (1:2, supernatant:isopropanol) at ambient temperature. The
stringy precipitate was further washed in isopropanol, allowed to stand in
acetone for an hour and then twice washed with fresh acetone. This
purified LBG was dried in air and then in a vacuum oven at 45 C.
Oxidation of Locust Bean Gum by Means of Galactose Oxidase
Purified LBG was dissolved at 0.1% (w/v) in 0.1M sodium phosphate (pH 7.0)
by heating to 80-90 C and periodically stirring with an Ultra-Turrax
homogeniser. Galactose oxidase (Sigma, G7907) was dissolved in sodium
phosphate, pH 7.0, to a concentration of 50 .mu.g/ml. A400 .mu.l aliquot
of this solution was added to 1.2 ml LBG solution (0.1% (w/v) and the
reaction mixture was incubated at 37 C for 5-16 hours, depending on the
particular experiment.
Conjugation of Glucose Oxidase (GOX) to Oxidised LBG
Glucose oxidase (Sigma product no.7141, 12.5 mg) was weighed into a tube
and 0.5 ml of oxidised LBG/galactose oxidase solution was added. The
mixture was tapped gently to dissolve the glucose oxidase, then stood at
ambient temperature for 2 hours. Sodium cyanoborohydride (NaBH.sub.3 CN,
10 .mu.l at 19 mg/ml) was added and the tube was left at ambient
temperature overnight. Glucose oxidase has an approximate molecular weight
of 160,000.
Assay for Active Conjugate (Cellulose Binding)
General Procedure
All assays exploited the ability of galactomannans to bind to cellulose by
polysaccharide:polysaccharide interaction. Sigmacell Type 20 cellulose
particles, with an average size of 20 m (from Sigma) were used to provide
a surface on which to capture cellulose binding molecules. A 50mg/ml
slurry of Sigmacell was made in PBS+Tween (0.05%) (PBST). The assays were
conducted in filter plates (Millipore, Product No. MAHVN4550) with 0.45 m
pore size filters. The plates were always pre-treated by soaking overnight
in PBST with bovine serum albumin (BSA, 2% w/v) to prevent non-specific
binding of enzyme or conjugate. Solutions were drawn through the filter
plate using a commercially available vacuum manifold (Anachem). The
overnight treatment solution (PBST/BSA) was removed prior to the
experiment. 100 .mu.l of the Sigmacell slurry (shaken immediately prior to
addition) was added to each well. 100 .mu.l of appropriately diluted
LBG-conjugates (see below) were added to the Sigmacell in the 96 well
plates, and incubated for 10 minutes to allow binding to the cellulose
surface. After 10 minutes the solution was drawn off under vacuum and the
cellulose washed by addition of 150 .mu.l of PBS+Tween (0.05%) five times.
The assays then continued as shown below for each of the conjugates.
Assay of LBG/GOX Conjugate
The stock LBG/GOX conjugate, containing 25 mg/ml glucose oxidase and 0.1%
(w/v) LBG, was diluted to a concentration of 25 .mu.g/ml in PBST +BSA (2%
wv). A control mixture containing 25 mg/ml glucose oxidase with unmodified
LBG was also diluted to the same concentration to check for non-specific
binding After incubation with the conjugates, the Sigmacell particles were
washed and then TMB GOx substrate was added to each well. The TMB
substrate was prepared by dissolving the following ingredients in 20 ml
water:
disodium phosphate (0.45 mg),
citric acid (150 mg),
D-glucose (0.54 g),
horse radish peroxidase (100ng) and
200 .mu.l of a 3,3',5,5'-tetramethylbenzidine (TMB) stock solution in DMSO
(100 mg/10 ml DMSO).
The substrate was left with the particles for several minutes, until a blue
colour developed. Before the optical densities were recorded, the
substrate solution was acidified by the addition of 50 .mu.l 2M HCl to
each well, and the yellow product was drawn through onto a flat bottom 96
well plate. The OD at 450 nm was determined and recorded on an automatic
plate reader.
Results
The reductive amination conjugation procedure yielded an active conjugate
of the two molecules, GOx and LBG, as shown by the optical density values
in Table 1. Both the cellulose binding activity of the galactomannan and
the enzymic activity of the GOx had been retained and had been effectively
combined in the conjugate. In contrast, the low OD value for the simple
mixture of LBG and GOx provides confirmation that conjugate performance
resulted from the chemical conjugation of the two molecules.
Table 1. Optical density values derived from the assay of test and control
samples produced through the reductive amination conjugation procedure.
Incubation with substrate was continued for only 5 minutes before the OD
values were determined.
TEST SAMPLE OPTICAL DENSITY
Conjugate of LBG/GOx 1.031
Mixture of LBG/GOx 0.001
EXAMPLE 2
Conjugation of Locust Bean Gum with Monoclonal Antibody 3299
Materials and Methods
LBG was purified and oxidised as described in Example 1.
Conjugation of Monoclonal Antibody 3299 to Oxidised LBG
A solution of a monoclonal antibody designated 3299 (MAb 3299, obtained
from Unipath), specific for the pregnancy hormone human chorionic
gonadotrophin (HCG), was dialysed overnight against 0.1M sodium phosphate
buffer, pH 6.5. The final concentration of antibody was adjusted to 10
mg/ml by further dilution with the same buffer, as appropriate. MAb 3299
has an appropriate molecular weight of 150,000.
A sample (20 .mu.l) of this MAb 3299 solution was mixed with 20 l of the
oxidised LBG solution (as above) and the mixture stood at room temperature
for 2 hours. A solution (1 .mu.l) of NaBH.sub.3 CN (19 mg/ml) was added
and the reaction left at ambient temperature, overnight.
Assay for LBG/Monoclonal Antibody 3299 Conjugate
LBG/MAb 3299 conjugate stock solution containing 5 mg/ml MAb 3299 and 0.05%
LBG was diluted in PBST+BSA (2% w/v) to an antibody concentration of 25
.mu.g/ml. A control mixture containing 25 mg/ml MAb3299 with unmodified
LBG was also diluted to the same concentration to check for non-specific
binding. After incubation with the cellulose particles and subsequent
washing, cellulose-bound monoclonal antibody was determined by means of
alkaline phosphatase conjugated tracer molecules. These were either (a)
rabbit anti-mouse IgG-alkaline phosphatase or (b) HCG-alkaline
phosphatase. These tracer conjugates were diluted in PBST +BSA (2% w/v).
a) Rabbit anti-mouse IgG-alkaline phosphatase tracer conjugate (Sigma) was
diluted 1/1000 and 150 .mu.l of this was added to each LBG/MAb cellulose
mix in the filter plates. After 1 hour the tracer conjugate was removed by
filtration and the plates washed 10.times. with 200 .mu.l per well PBST.
Alkaline phosphatase substrate (Sigma 104) was added to each well (200
.mu.l para-nitrophenyl phosphate, pNPP, lmg/ml in diethanolamine buffer,
pH 10) and maintained at ambient temperature until a significant yellow
colour had developed (about 30 minutes). The yellow product was then drawn
through onto a microtitre plate in the usual way, and the OD at 405 nm
determined.
b) HCG-Alkaline phosphatase tracer conjugate was diluted 1/200 and 150
.mu.l of this was added to each LBG/MAb cellulose mix in the filter
plates. The experiment then proceeded as in (a) above.
Results
The reductive amination procedure was found to be effective for conjugating
antibodies to LBG, as shown by the values in Table 2. As for GOx, the
values show that a chemical cross-linking process was necessary (rather
than simple mixing) to make an active conjugate, and the procedure did not
result in loss of binding activity in either molecule. The different
tracer molecules (anti-mouse IgG and HCG) confirmed that the antibody had
bound to the cellulose in a form that retained its specific, immunological
binding ability (HCG), as well as its general immunoglobulin qualities
(anti-mouse IgG).
The low but measurable binding values found with the mixed sample is
evidence of a general "stickiness" associated with the monoclonal antibody
and cellulose particles (non-specific adsorption). However, the difference
between the two sets is clear evidence of acceptable conjugation
efficiency.
Table 2. Optical density values derived from the assay of test and control
samples produced through the reductive amination conjugation procedure.
OD 405 nm
TEST SAMPLE a) - mouse IgG-AP b) HCG-AP
Conjugate of LBG/MAb 3299 1.040 0.831
Mixture of LBG + MAb3299 0.063 0.071
EXAMPLE 3
Conjugation of Locust Bean Gum with scFv3299
Materials and Methods
LBG was purified and oxidised as described in Example 1.
The single chain Fv antibody fragment of 3299 (scFv3299) is a genetically
engineered fragment consisting of the variable regions of the 3299 parent
antibody, produced in micro-organisms transformed with the relevant genes,
suitably formatted for that host. To work with this material it was first
necessary to culture the transformed host organism, induce expression of
the gene and then purify the scFv from the supernatant fluid of the
culture. The engineered scFv3299 also carried a short oligo-histidine
"tail" which could bind to immobilised nickel ions as a means of
purification (the IMMAC procedure). scFv 3299 has an approximate molecular
weight of 26,000.
Production and Purification of ScFv
The antibody fragment scFv3299, modified by the addition of a short
oligo-histidine tail, was produced in a transformed host micro-organism
(e.g. E. coli) using conventional methods well known to those skilled in
the art. The scFv3299 protein was purified by standard methods through an
IMMAC procedure and then through Mono S (cation exchange) chromatography.
The purified protein solution was subjected to a buffer-exchange process
through a PD-10 gel filtration column (Pharmacia) into 0.1M phosphate
buffer pH 6.5.
Thiolation of ScFv3299 with 5-Acetylmercaptosuccinic Anhydride (SAMSA)
Purified ScFv 3299 at 1.4 mg/ml in phosphate buffer pH 6.5 0.1M (150 .mu.l)
was placed in a 0.6 ml "Reactivial" (Pierce). A solution (20 .mu.l) of
SAMSA (supplied by Sigma) dissolved in dimethylformamide at the rate of 6
mg/ml was then added to the vial. This reaction mixture was stirred for 30
min at ambient temperature. The following were then added in succession,
the resultant mixture being stirred for 5 minutes after each addition:
25 .mu.l 0.1M EDTA, pH 8.0
100 .mu.l 0.1M Tris HCl pH 7.0
100 .mu.l 1M Hydroxylamine
After the final addition, the mixture was diluted to 2.5 ml in 0.1M sodium
phosphate+5 mM EDTA, pH 6.5 and applied to a PD-10 gel filtration column.
The protein-thiol conjugate was eluted in 3 ml phosphate +EDTA buffer,
which was concentrated again by reducing the volume to 150 .mu.l through a
Centricon 10 device (Amicon).
Derivatisation of LBG with MPBH
4 (4-N-maleimidophenyl) butyric acid hydrazide.HCl (MPBH, Pierce Product No
22305) was dissolved at 10 mg/ml in dimethylsulphoxide (DMSO). Oxidised
LBG was prepared as described above, except that at the end of the process
the galactose oxidase left with the product was denatured by heating to
98.degree. C. for 15 mins. The resultant solution was subjected to buffer
exchange with 0.1M sodium acetate, pH 5.5, by means of a Centricon 30
device (Amicon) and then the volume was adjusted back to the starting
volume by the addition of more buffer solution, as appropriate. At this
point in the procedure, MPBH in DMSO (21.3 .mu.l) was added to 600 .mu.l
of the oxidised LBG solution, to give a final MPBH concentration of lmM
This reaction mixture was kept at ambient temperature for 2 hours with
gentle agitation. The derivatised LBG product was then subjected to buffer
exchange with O.lM phosphate buffer, pH 6.5, by means of a Centricon 30
device, after which the volume was readjusted to 600 .mu.l.
Conjugation of SAMSA-Derivatised ScFv with MPBH-Derivatised LBG
The ScFv-SAMSA solution (75 .mu.l containing 0.105 mg) was mixed with
LBG-MPBH (25 .mu.l) and the volume made up to 125 .mu.l with phosphate
buffer. The reaction mixture was left at ambient temperature overnight.
Assay for LBG/scFv3299 Conjugate
LBG/scFv3299 conjugate stock solution containing 0.34 mg/ml scFv3299 and
0.02% LBG was diluted in PBST+BSA (2% w/v) to an scFv concentration of 34
.mu.g. As with the whole antibody conjugate, a mix of ScFv and LBG;
containing the same relative amounts, were diluted to the same
concentration. The assay procedure was as described above for the whole
antibody conjugate, but only with the HCG-alkaline phosphatase tracer
conjugate (since an scFv fragment could not be detected with anti-mouse
IgG).
Results
The scFv3299 antibody fragment was found to have conjugated with LBG, as
shown from the values in Table 3, even though a different, more complex
conjugation method had been used. The difference between the control
mixture values and the conjugate values is less than with the other
systems, indicating either a lower efficiency of conjugation or a greater
tendency for the unconjugated material to adsorb non-specifically. A lower
conjugation efficiency is more likely, since a longer incubation time with
substrate was needed to produce adequate OD values.
However, the results clearly show that even with the scFv fragment, a
significant and useful degree of chemical conjugation had been achieved.
Table 3. Optical density values derived from the assay of test and control
samples produced through the SAMSA/MPBH conjugation procedure.
TEST SAMPLE OPTICAL DENSITY (405 nm)
Conjugate of 0.714
LBG/scFv3299
Mixture of LBG + 0.147
scFv3299
EXAMPLE 4
Conjugation of Active Proteins to Tamarind Seed Xyloglucan (TXG)
Tamarind seed polysaccharide (Glyoid 3S from Dainippon Pharmaceutical Co.,
Osaka, Japan) was dissolved in deionised water (16 h at 25 C or 10 min at
80 C followed by 2-3 h at 25 C) to give a 0.5-1.0% w/v suspension which
was clarified by centrifugation (20,000 g, 30 min.), dialysed extensively
against deionised water, and lyophilised.
The resulting TXG was then formed into conjugates with glucose oxidase, MAb
3299 and scFv 3299 exactly as described in Examples 1, 2 and 3 and the
resulting conjugates were found to bind to cellulose.
EXAMPLE 5
Binding of Locust Bean Gum and Tamarind Seed Xyloglucan (TXG) to Benefit
Agents Carried in Particles for Targeting to Fabric
In this example, a blue dye was used as an example of a small organic
molecule (i.e. a "model" benefit agent), silica was used as an example of
a porous particle, and cotton was used as an example of a
cellulose-containing fabric surface.
Materials and Methods
Preparation of Particles
Porous silica particles (mean pore-size 2 nm, average particle size 9 m)
were obtained from Joseph Crosfield & Sons (Warrington U.K.) Approx. 25 mg
of silica was placed in each of three round-bottomed plastic tubes. 0.5 ml
of 0.4% Coomassie blue (in purified water) was added to each and then they
were mixed thoroughly and left for 1 hour at ambient temperature.
The tubes were centrifuged for 5 minutes [13,000 r.p.m. in a
microcentrifuge (MSE)] and the supernatants removed. Each of the three
tubes then received a different treatment in which 0.5 ml of one of the
following was added.
a) purified water.
b) 0.1% TXG (ex. Tamarind seed) in phosphate buffer, pH 7, prepared as
described in Example 4.
c) 0.1% galactomannan (ex. Iocust bean, LBG) in phosphate buffer, pH 7,
prepared as described in Example 1.
The three tubes were mixed thoroughly and then rotated overnight at ambient
temperature. After the overnight treatment, the tubes were centrifuged as
before and the supernatants removed. The particles were washed once in
water and then twice in saline (pH 7).
The particles were then resuspended in 1 ml saline (pH 7) and left at 4 C
until required. There were, therefore, three different silica slurries,
each containing approx. 25 mg silica in 1 ml of saline. The slurries were
designated a), b), and c), depending on the treatment received, as set out
above.
Targeting the Particles to Fabric
Three squares (approximately 1 cm.times.1 cm) were cut from white cotton
cloth. These were equilibrated in saline, pH7, by shaking each one with 2
ml of saline in a plastic tube. There were then three tubes each
containing 2 ml of saline and one cotton square.
To each tube 100 .mu.l of silica slurry was added. A different sample of
slurry was added to each tube: either a), b), or c). The three tubes were
rotated slowly at room temperature for 2 hours, after which the three
cotton squares were removed and photographed. The appearance of blue
coloration was carefully observed and noted.
Results
Treatment with the polysaccharides had endowed the particles with an
ability to bind to the fabric. Moreover the Coomassie blue had been
retained in the pores of the silica, the retention being aided by the
polysaccharide coating. After the gentle but significant exposure and
washing process (rotation for 2 hours), colour was found to adhere to the
fabric surface, as shown in Table 4.
Table 4. Blue coloration remaining on fabric squares treated with Coomassie
blue loaded silica particles coated with LBG or TXG.
PARTICLES RESULTING
APPLIED TO COTTON COLOUR OF SQUARE
slurry a) white
slurry b) medium blue
slurry c) dark blue
It is concluded that the LBG and TXG are able to adsorb in some way to the
particle surface and yet still retain the ability to bind to cellulose,
providing the means for particle targeting to cellulose surfaces.
EXAMPLE 6
Binding of Locust Bean Gum (LBG) to Fragrance-Enriched Particles for
Targeting to Fabric.
Materials and Methods
Preparation of Particles
Porous silica particles (mean pore-size 2 nm, average particle size 9 m)
were obtained from Joseph Crosfield & Sons (Warrington U.K.).
Approximately 25 mg of silica was placed in each of two round-bottom
plastics tubes. 0.5 ml of the fragrance florocyclene (obtained from Quest
International, Ashford U.K.) was added to each of the tubes and mixed
thoroughly. The lids of the tubes were sealed to prevent evaporation of
the fragrance.
The two tubes were rotated overnight with a gentle tumbling motion to allow
the fragrance molecules to enter the pores of the particles. This was done
at ambient temperature.
The tubes were centrifuged for 5 minutes [13,000 r.p.m. in a
microcentrifuge (MSE)] and the supernatants discarded. Each of the two
tubes then received a different treatment in which 1 ml of one of the
following was added.
a) 1 ml of 0.1% LBG (Sigma Product No. G-0753)
b) 1 ml of purified water.
The two tubes were mixed thoroughly and then rotated overnight at ambient
temperature. After the overnight treatment, the tubes were centrifuged as
before and the supernatants removed. The particles were washed once in
water and then twice in saline (pH 7). The particles were then resuspended
in 1 ml saline (pH 7) and left at ambient temperature until required.
There were therefore two different slurries, each containing approximately
25 mg of silica in 1 ml of saline. The slurries were designated a) or b)
depending on the treatment received as set out above.
Targeting the Particles to fabric
Two squares (approximately 1 cm.times.1 cm) were cut from white cotton
cloth.
2 ml of saline was added to each of two plastics tubes. 100 .mu.l of slurry
a) was added to one; 100 .mu.l of slurry b) was added to the other. One
cotton square was added to each of the two tubes a) and b).
The two tubes were rotated slowly at room temperature for two hours; after
which the cotton squares were removed with tweezers. Each square was
placed in a separate Petri dish. The lids of the Petri dishes were used to
prevent evaporation of the fragrance.
Results
Eight people were asked to compare cotton squares treated with slurry a)
and slurry b) and decide which of the cotton squares smells the strongest.
6 out of 8 said that a) was the strongest
1 out of 8 said that b) was the strongest
1 out of 8 could not tell the difference.
It was concluded that the cotton square treated with slurry a) had more
fragrance on it due to the targeting effect of the LBG.
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